Pta LDHA double mutant Escherichia coli SS373 and the method of producing succinic acid therefrom

ABSTRACT

This invention relates to a mutant  Escherichia coli  SS373 and the production of succinic acid by using the above strain. In detail, a novel  E. coli  SS373 (W3110 pta::Tn10 ldhA::Km) with the deficiency in the acetate and lactate forming pathways was constructed by genetic engineering technique. An aerobically grown SS373 was then cultured by means of the anaerobic condition shift during the succinate producing stage, which resulted in the efficient production of succinic acid with a higher yield.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a mutant Escherichia coli SS373 and theproduction of succinic acid by using the above strain. In detail, anovel E. coli SS373 (W3110 pta::Tn10 ldhA::Km) with the deficiency inthe acetate and lactate forming pathways was constructed by geneticengineering technique. An aerobically grown SS373 was then cultured bymeans of the anaerobic condition shift during the succinate producingstage, which resulted in the efficient production of succinic acid witha higher yield.

2. Description of the Prior Art

Succinate is one of the basic metabolites and an intermediate in the TCAcycle of the biological system. In the petrochemical industry, succinateserves a precursor of 1,4-butandiol. tetrahydrofuran, γ-butyrolactone.It is also useful as an ingredient in the food and cosmetic industry.Succinic acid is commercially produced by a chemical process. Recentlythe biological process has been of interest for an environmentally cleanprocess. In addition. the biological process could produce succinatefrom low-cost renewable resources. For the reasons of as above, thebiological succinate production has been intensely studied in the recentyears. Among these studies, strict anaerobic Anaerobiospirillumsucciniciproducens has been particularly well examined (U.S. Pat. Nos.5,573,931, 5,521,075, 5,504,004). A. succiniciproducens, however, has acomplex nutrient requirement and slows growth rate as well as difficultyin the production process associated with the strict anaerobe.

SUMMARY OF THE INVENTION

To solve the problems of a strict anaerobe in the succinate production,a facultative anaerobic E. coli was genetically engineered. By using themutated E. coli, the succinate production with higher yield wasachieved. Therefore, the objective of the invention herein is theconstruction of a mutant E. coli and enhanced production of succinate byusing the mutant E. coli.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents the metabolic pathway of SS373 based on various carbonsources.

FIG. 2 indicates the succinate production profile by SS373.

DETAILED DESCRIPTION OF THE INVENTION

The invention herein is characterized by Escherichia coli SS373(W3110pta::Tn10 ldhA::Km). The method of the anaerobic production of succinateafter aerobic growth of cells is also involved. The detaileddescriptions are as follows:

As reported in the Bergey's Manual, E. coli has the followingcharacteristics: facultative anaerobe, rod shaped, Gram-positive, simplenutrient demand, fast growth rate (doubling time≈20 min.), temperatureoptimum of 37° C., and pH optimum of 7.0. Especially, E. coli yields amixture of acetate, lactate, formate, succinate, and ethanol fromglucose in the anaerobic condition. The physiology and genetics of E.coli have been well studied, and the E. coli metabolism could be easilycontrolled and estimated. In addition, the metabolic engineering couldbe readily applied by means of genetic engineering technique.

Principle of Succinate Mass-production

To produce succinate by using E. coli, an E. coli W3110 was modifiedgenetically. The modified E. coli was further optimized to lead to anenhanced production.

Because E. coli carries out a mixed acid-fermentation, the metabolicpathway of E. coli should be altered to efficiently produce succinate.By means of the genetic block of the pathway involved in the otherproducts. the succinate production would be improved. At first the genesof pta and ldhA of E. coli, which encode the first enzyme of acetate andlactate pathway, were mutated.

The constructed strain was cultured in an aerobic condition with highgrowth rate which in turn produced succinate in the anaerobic condition.The succinate therefrom is able to penetrate the cell membrane toaccumulate in the medium which in turn prevents a feedback control ofcells. The accumulated succinate can be recovered with high purity byelectrodialysis technique (Hongo, M., Appl. Environ. Microbiol. 52-2314-319 (1986)).

Construction of a Double Mutated E. coli

Construction of a double mutated E. coli was carried out by the methodsuggested by Silhavy.

Step 1. Preparation of Transformed P1 Phage

P1 lysates of a E. coli CP993 (pta::Tn10-lacZ1)(Shin, S. A. and C. K.Park. J. Bacteriol., 177, 4696-4702 (1995)) and a E. coli NZN117(ldhA::Km)(Bunch. P. K. et al. Microbiology, 143, 187-195 (1997)) wereprepared respectively.

Step 2. P1 Transduction of the pta::Tn10-lacZ1 to W3110

An E. coli W3110 (E. coli genetic stock center collection number (CGSC)4474) was used as a recipient strain. The insertion mutated gene(pta::Tn10-lacZ-1) of E. coli CP993was transferred to E. coli W3110 byP1 transduction. The mutant E. coli strains were selected on thetetracyclin selection plate which yielded an E. coli W3110pta::Tn10-lacZ-1

Step 3. P1 Transduction of the ldhA::Km to W3110pta::Tn10-lacZ-1

To obtain a lactate-production deficient strain. P1 lysate of NZN1117was infected with E. coli W3110 pta::Tn10-lacZ-1. The selected strain onthe kanamycin plate was an double mutated W3110 pta::Tn10-lacZ-1ldhA::Km.

Principle of Succinate Production in SS373 from Various Carbon-sources

Though the pathways to succinate slightly differ from one anotherdepending on the carbon source, the phosphorylation is a common process(FIG. 1). In the case of glucose, which is the most common carbonsource, the main phosphate donor has been known to bephosphoenolpyruvate (PEP) when glucose is transported by phosphatetransferase system (PTS). The PEP involved in the glucose uptakeconverts to pyruvate, and the chance for the succinate production isrelatively reduced because succinate is derived from oxaloacetate (OAA).Hence, the phosphate groups are delivered from ATP in the cases ofgalactose, xylose, and maltose, the PEP would be saved as compared withthat of the case with glucose. The conservation of PEP, which serves asa phosphate donor in the PTS, would lead to the increase of succinateproduction as well as a decrease of by-product formation.

This invention will be described detail in the following examples but isnot limited thereby.

EXAMPLE 1

Construction of a Double Mutated E. coli for the Succinate Production

Step 1. Preparation of Transformed P1 Phage

The P1 transduction was carried out by the Silhavy method. Each E. colistrain of pta::Tn10-lacZ-1 and ldhA::Km was pre-cultured in 3ml of TGCmedia (0.1% glucose, lacto-tryptone, 10 mM CaCl₂). The overnight growncells were transferred to the 3 ml of TGC media and cultured for 1 hr at35° C. in a shaking incubator. When the absorbance (600 nm) of cells wasreached at 0.1, the P1 phage (30 μl in the concentration of 1010 pfu/ml)was infected and cultured for 2-3 hrs. After the cell lysis, chloroform(0 ml was added and then supernatant was prepared by centrifuge. Thesupernatant

Step 2. P1 Transduction of the pta::Tn10-lacZ1 to W3110

The overnight grown E. coli W3110 was prepared by centrifuge. After thedispersion of cells with 0.5 ml of divalent ion solution (10 mM MgSO₄, 5mM CaCl₂), the P1 lysate of pta::Tn10-lacZ-1 (0.01-0.1 ml) was appended.The mixture was left to stand for 15 minutes at room temperature. Thecells were collected by centrifuge and then washed twice by 1 ml of 1Msodium citrate. After the activation in LB medium, the mutant cells wereselected on the LB-agar plate containing tetracycline (13 μg/ml).

Step 3. P1 Transduction of the ldhA::Km to W3110 pta::Tn10-lacZ-1

The P1 lysate of ldhA::Km from step 1 was infected to the strainobtained from step 2. After the same procedure of step 2, a doublemutant of W3110 pta::Tn10-lacZ-1 ldhA::Km was obtained on the LB-agarplate containing kanamycin (20 μg/ml).

The finally obtained E. coli W3110 pta::Tn10-lacZ-1 ldhA::Km was namedE. coli SS373. The E. coli SS373 was deposited on the 28th of Jul. 1997in the Korea Collection of Type Culture(KCTC; 52, Ereun-dong, Yusong-ku,Taejeon 305-333 Republic of Korea), which is an international straindeposit institute by ale Budapest Convention, and the deposit number wasassigned as KCTC 8818P. For the purpose of PCT internationalapplication, a conversion of the original deposit under the BudapestTreaty was made on Jul. 29, 1998, and a new deposit number was obtainede.g. KCTC 0506BP.

The E. coli SS373 could be cultured on a glucose medium in an anaerobiccondition because it could produce acetyl-CoA while E. coli NZN111(ClarkD. P. FEMS Microbiol Rev., 63, 223-234 (1989)) could not.

EXAMPLE 2

Succinate Production in the Glucose Medium

The E. coli SS373 was cultured on a glucose-based medium. The componentsof medium were represented as Table 1.

TABLE 1 Na₂HPO₄ · Yeast Component Glucose H₂O NaH₂PO₄ Extract Na₂CO₃Concentration 15 7 3 5 3.18 (g/l) Note:The pH was pre-set to 7.0 byadding a few drops of conc. H₂SO₄.

A single colony of SS373 was sub-cultured in a 15 ml test tube at 37° C.for 12 hours. Cells were transferred to a 50 ml medium in 250 mlErlenmeyer flask and cultured until absorbance reached 0.5 at 600 nm.The actively grown cells from above were inoculated to a 2.5-literjar-fermentor containing 1-liter medium and cultured at 37° C., pH 7.0in aerobic condition (350 rpm, 1 vvm). When the absorbance(600 nm)reached 4.0, aeration was stopped and mixed gas (5% CO₂, 95% N₂) wasfluxed in. Upon shifting to anaerobic conditions, 500 ml of a glucosesolution (60 g/l) was added. Thereafter, 11 g/l of succinate wasproduced with 0.8 g/l of pyruvate in 34 hours of culture. (FIG. 2)

The concentrations of succinate and pyruvate were estimated by using aHPLC-UV system (Gilson, France) with carbohydrate analysis column(HPX-87H, Bio-Rad). The glucose concentration was measured by theGlucose-Analyzer (2300STAT, Yellow Spring Instruments).

EXAMPLE 3

Succinate Productions Based on Various Carbohydrates

The E. coli SS373 and W3110 were cultured in the media containingdifferent carbon sources (Table 2). The carbon sources used wereglucose, galactose, maltose, and xylose, respectively.

TABLE 2 *Carbon Na₂HPO₄ · Yeast Component source H₂O NaH₂PO₄ ExtractNa₂CO₃ Concentration(g/l) 10 7 3 5 3.18 Note:The pH was set to 7.0 byadding a conc. H₂SO₄. *Carbon sources were glucose, galactose, maltose,and xylose, respectively.

A single colony of SS373 was sub-cultured in a 15 ml test tube at 37° C.for 12 hours. Cells were transferred to a 10 ml medium in 100 mlErlenmeyer flask. The biomass was set to an approximate absorbance of1.0 at 600 nm. The flask was flushed with 5% CO, gas and sealed by usinga silicon stopper to maintain anaerobic condition. Cells were culturedfor 8 hrs at 37° C. and organic acids formation were investigated (Table3).

In the cases of wild strains, e.g., W3110, the major organic acids werelactate and acetate, while succinate and pyruvate were the major factorsin the SS373. In the SS373, the proportions of succinate to pyruvatewere varied depending on the carbon sources used. The glucose mediumshowed 1:2 of succinate to pyruvate with 1:0.8 for maltose and 1:0.3 forgalactose and xylose. Nearly pure succinate was obtained in theconcentration of 1.9 and 1.6 g/l from galactose and xylose,respectively. Therefore, the use of non-PTS carbohydrates was preferablein producing succinate with high purity and yield because PEP used inphosphorylation was conserved

TABLE 3 Effect of Carbon Sources on the Succinate Production in E.coliSS373 Carbon Succinate Pyruvate Lactate Acetate Strain source (g/l)(g/l) (g/l) (g/l) W3110 Glucose 0.6 0 1.0 1.0 Maltose 0.5 0 1.6 1.6Galactose 2.1 0 0.8 2.3 Xylose 1.6 0.2 0.9 1.6 SS373 Glucose 2.7 5.3 00.6 Maltose 2.3 1.8 0 0.1 Galactose 1.9 0.6 0 0.3 Xylose 1.6 0.4 0 0.4

As noted, succinate in a novel E. coli SS373 could be produced with lesseffort to maintain strict anaerobic condition and without complexnutrient supply. In addition, E. coli SS373 showed fast growth rate dueto the efficient succinate production. Moreover, nearly pure succinatecould be produced by using a carbon source with the result of conservingPEP.

What is claimed is:
 1. A phosphotransacetylase (pta) lactatedehydrogenase (ldhA) double mutant E. coli SS373 (KCTC 0506BP) whichlacks the ability to produce lactate and acetate.
 2. A method ofproducing succinic acid comprising the steps of: (a) culturing the ptaldhA double mutant E coli SS373 of claim 1 via a two-stage culturewherein E. coli SS373 is initially cultured under aerobic conditions forbacterial growth, and is subsequently cultured under anaerobicconditions for succinic acid production; and (b) recovering succinicacid from the culture medium.
 3. The method of producing succinic acidaccording to claim 2, wherein said two-stage culture is performed in thepresence of a substrate which does not require phosphoenolpyruvate formembrane transport.
 4. A method of making a succinic acid-producing E.coli strain, wherein insertional mutations in the pta and ldhA genesresult in a strain deficient in the production of lactate and acetate,and enhanced production of succinic acid.